The latency associated transcripts (LATs) are the only viral gene products detected in neurons latently infected with herpes simplex virus (HSV). Moreover, detection of the virus in latent neurons has previously depended upon detection of LAT. Two central questions in HSV pathogenesis are (i) in latently infected animals, how many neurons actually harbor virus and (ii) what role, if any, does the LAT region play in the establishment, maintenance and reactivation of virus from latent infection and what is the LAT mechanism of action. The goal of this project is to help answer these questions by using a combined in vivo and in vitro approach. With in vivo studies, we will determine the true number of neurons in mice latently infected with either HSV wild type or LAT region mutants, and if this number changes as a function of time throughout the latent phase of infection. This will be done by using an in situ polymerase chain reaction (PCR) to detect HSV DNA in tissues derived from latently infected animals. In this way, the presence of HSV is detected in a manner independent of virus gene expression. We have also developed an in vitro model of HSV of acute and long term neuronal infection: nerve growth factor [NGF] differentiated Pcl2 cells. This system will be tested for relevancy by a range of biochemical tests and, if validated, used to examine: (i) the precise lytic phase defects, if any, of LAT region mutants, and (ii) if these defects relate to the in vivo properties of the virus. In addition, long term infected NGF treated PCl2 cells, which have been shown to reactivate HSV following NGF removal, will be used to provide information about HSV latent infection which is difficult to obtain with in vivo analysis, such as: (i) the physical state of the virus chromosome during quesicent infection as well as (ii) the first viral and host functions expressed in wild type and LAT region mutant long term infected PC12 cells, following release from NGF. These results will provide clues as to what viral and host factors are involved in managing or participating in the reactivation from latency and will help guide the in vivo work described in Project 2. Overall, therefore, the in vivo experiments, conducted in cooperation with Project 1, will determine the if the LAT region is involved in influencing the establishment or maintenance of the latent state, allowing for rationale hypotheses of LAT function. The in vitro studies will determine both the lytic phase behavior of LAT region mutants as well explore the relevancy of an exciting tissue culture model of latency or "long term" HSV infection of neurons.